Preparation and value method of alpha-synuclein standard material

Abstract

The present application relates to the technical field of biometrics and in vitro diagnosis standardization, and particularly relates to a preparation and value determination method of alpha-synuclein solution standard substance, comprising the following steps: cloning a gene coding alpha-synuclein into a prokaryotic expression vector for recombinant expression, purifying the protein raw material with purity greater than or equal to 99% through affinity chromatography and ion exchange chromatography; diluting and sub-packaging the protein raw material for storage to obtain the solution standard substance; determining the value of the standard substance by using an isotope dilution mass spectrometry method based on amino acid analysis, calculating the mass concentration value by measuring the peak area ratio of stable amino acids and isotope-labeled amino acids after hydrolysis, and tracing the value to the international unit system through the amino acid primary standard substance; and performing uniformity testing and stability investigation on the standard substance, solving the problem of lack of standard substance with metrological traceability in the alpha-synuclein detection field, and obtaining the standard substance which can be used for value transmission and detection result standardization among different in vitro diagnosis platforms.

Description

Technical Field

[0001] This invention relates to the field of biometrics and in vitro diagnostic standardization technology, specifically to the preparation and determination method of α-synuclein solution standard material. Background Technology

[0002] α-Synuclein (α-Syn) is a presynaptic protein of neurons composed of 140 amino acids. Under physiological conditions, it exists as a soluble, unfolded monomer and participates in regulating synaptic plasticity, neurotransmitter release, and vesicle transport. Under pathological conditions, α-Syn misfolds, forming β-sheet-rich oligomeric intermediates, which further aggregate into insoluble amyloid filaments, eventually depositing to form Lewy bodies and Lewy neurites. This process is a core molecular pathological event in synucleinogenic disorders such as Parkinson's disease, Lewy body dementia, and multiple system atrophy.

[0003] In recent years, in vitro diagnostic technologies based on α-Syn and its derivatives in cerebrospinal fluid and peripheral blood have developed rapidly, providing new tools for early screening, differential diagnosis, and disease monitoring of neurodegenerative diseases. In particular, detection methods based on α-Syn seed amplification technology have shown extremely high diagnostic specificity. In addition, immunological detection methods for different forms of α-Syn are also being continuously developed.

[0004] However, a key challenge in the field of α-Syn in vitro diagnostics is the standardization of test results. Due to the lack of internationally recognized, metrologically traceable α-Syn protein standard materials, different research institutions and diagnostic companies use their own prepared recombinant proteins or commercially sourced proteins as calibrators. These internal standards vary greatly in purity, conformation, aggregation state, and accuracy of value determination, making it impossible to directly compare test results between different platforms and laboratories. Cutoff values ​​are difficult to unify, which seriously hinders the standardization of the clinical translation and application of related biomarkers.

[0005] In existing technologies, conventional methods for determining protein concentration mainly rely on relative methods such as ultraviolet spectrophotometry or BCA colorimetry. However, α-Syn, as an inherently disordered protein, lacks a stable tertiary structure, and its extinction coefficient is significantly affected by solution conditions. Concentration values ​​determined by the aforementioned relative methods cannot be traced back to the International System of Units (SI), and significant systematic biases exist between different batches and laboratories. Therefore, there is an urgent need to establish an accurate and traceable α-Syn protein standard based on absolute quantitative methods, along with its preparation and determination methods. Summary of the Invention

[0006] The technical problem to be solved by this invention is: how to provide a method for preparing and determining the value of α-synuclein solution standard substances, so that the obtained standard substances have the characteristics of high purity, accurate values, traceability to the International System of Units, and can be used as primary calibrators for the transfer of values ​​and standardization of test results between different in vitro diagnostic platforms.

[0007] To address the aforementioned technical problems, this invention provides a method for preparing and determining the value of α-synuclein standard substances, comprising the following steps: Step 1, Protein Preparation: The gene encoding α-synuclein is cloned into a prokaryotic expression vector for recombinant expression. Soluble components containing the α-synuclein are collected and purified by affinity chromatography and ion exchange chromatography to obtain α-synuclein raw material with a purity ≥99%.

[0008] Step 2, Standard Substance Dispensing: After diluting the α-synuclein raw material to the target concentration, dispense it into cryovials and store at -80°C to -60°C to obtain α-synuclein solution standard substance.

[0009] Step 3, Value Assignment: The standard substance is assigned a value using isotope dilution mass spectrometry based on amino acid analysis. The value assignment method includes: mixing the standard substance with isotope-labeled amino acids and then performing acid hydrolysis. At least two free amino acids that are stable under acid hydrolysis conditions are used as quantitative targets. The peak area ratio of each amino acid to the corresponding isotope-labeled amino acid is determined by liquid chromatography-mass spectrometry, and the mass concentration value of the standard substance is calculated. The mass concentration value is traced back to the International System of Units (SI) through the primary amino acid standard substance.

[0010] Step 4: Quality Evaluation: Conduct homogeneity testing and stability assessment on the standard substance to confirm that its homogeneity and stability meet the technical specifications of the standard substance.

[0011] Furthermore, in step one, the expression vector carries a histidine tag, the affinity chromatography is nickel ion metal chelate affinity chromatography, and the ion exchange chromatography is anion exchange chromatography.

[0012] Furthermore, in step one, the anion exchange chromatography utilizes the acidic isoelectric point characteristics of α-synuclein and employs a DEAE column for purification to remove charge variant impurities.

[0013] Furthermore, in step three, the stable free amino acid is selected from at least two of valine, phenylalanine, and isoleucine.

[0014] Furthermore, in step three, the acid hydrolysis conditions are hydrolysis in 6 mol / L hydrochloric acid solution at 108°C to 112°C for 50 to 70 hours.

[0015] Furthermore, in step three, the isotope dilution mass spectrometry method uses the bracket method for quantitative calculation, and the matrix effect is eliminated by encapsulating the sample to be tested with high-standard and low-standard solutions.

[0016] Furthermore, step one also includes characterizing the purity of the purified α-synuclein raw material using at least two of the following methods: gel size exclusion chromatography, reversed-phase high-performance liquid chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0017] Furthermore, in step four, the stability study includes short-term stability studies under at least two different temperature conditions, long-term stability studies under conditions ranging from -80°C to -60°C, and fusion stability studies.

[0018] Furthermore, in step three, the setting of values ​​is performed by at least two independent operators, and the set value data is sequentially subjected to outlier testing, normality testing, and mean consistency testing.

[0019] Furthermore, the amino acid sequence of the α-synuclein is shown in SEQ ID NO:1.

[0020] Compared with the prior art, the present invention has the following advantages: (1) This invention uses a two-step purification strategy of affinity chromatography and ion exchange chromatography, combined with purity characterization by three orthogonal methods: gel size exclusion chromatography, reversed-phase high performance liquid chromatography and SDS-PAGE, to obtain α-synuclein raw materials with a purity of ≥99%, providing a high-quality material basis for subsequent accurate determination.

[0021] (2) The present invention uses isotope dilution mass spectrometry based on amino acid analysis to perform absolute quantification at the level of free amino acids after complete hydrolysis of protein. It selects amino acids that are stable under acid hydrolysis conditions as the quantification object, and eliminates matrix effects by using bracket method to calculate and realize direct traceability of standard substance mass concentration values ​​to the International System of Units. It eliminates the systematic bias caused by protein conformational changes in traditional relative methods such as ultraviolet spectrophotometry and BCA method.

[0022] (3) The present invention establishes a complete standard material quality evaluation system, including homogeneity test, short-term stability test under multiple temperature conditions, long-term stability test and remelting stability test, as well as a complete statistical analysis process of independent two-person value determination, outlier test, normality test and consistency test, which ensures the metrological reliability of the standard material value determination results. Attached Figure Description

[0023] Figure 1 This is an α-synuclein sequence.

[0024] Figure 2 This is the sequence of the recombinantly expressed α-synuclein.

[0025] Figure 3 This is an SDS-PAGE electrophoresis image of α-Syn protein.

[0026] Figure 4 The image shows a high-performance liquid chromatography (HPLC) chromatogram of the raw material for a 1 mg / mL α-Syn protein standard.

[0027] Figure 5 The image shows a high-performance liquid chromatography (HPLC) chromatogram of a 5 mg / mL α-Syn protein standard sample.

[0028] Figure 6 The high-performance liquid chromatography (HPLC) chromatogram of the raw material for the 5 mg / mL α-Syn protein standard (partial magnification).

[0029] Figure 7 The reverse high performance liquid chromatogram of raw material C8 for 5 mg / mL α-Syn protein standard is shown.

[0030] Figure 8 The reversed-phase high-performance liquid chromatogram (partial magnification) of the raw material C8 for the 5 mg / mL α-Syn protein standard.

[0031] Figure 9 The image shows the MALDI-TOF mass spectrum of the α-Syn protein trypsin digestion solution.

[0032] Figure 10 This is a high-resolution mass spectrum of the α-Syn protein.

[0033] Figure 11 Photograph of the aliquoted α-Syn protein solution standard.

[0034] Figure 12 The figure shows the optimization of hydrolysis time for α-Syn protein samples.

[0035] Figure 13 The stability of the α-Syn protein solution standard was investigated at 4°C.

[0036] Figure 14 Chromatogram of the stability monitoring of α-Syn protein solution standard material stored at 4°C for 7 days.

[0037] Figure 15 To investigate the stability of α-Syn protein solution standard material at room temperature.

[0038] Figure 16 Chromatogram of the stability monitoring of α-Syn protein solution standard material stored at room temperature for 7 days.

[0039] Figure 17 To investigate the stability of α-Syn protein at room temperature for 7 days.

[0040] Figure 18 To investigate the stability of α-Syn protein solution standard at 40°C.

[0041] Figure 19 Chromatogram of the stability monitoring of α-Syn protein solution standard material stored at 40°C for 7 days.

[0042] Figure 20 Chromatogram of the stability monitoring of α-Syn protein solution standard material stored at -80°C for 2 months.

[0043] Figure 21 The chromatogram is a result of repeated freeze-thaw cycles of the α-Syn protein solution standard.

[0044] Figure 22 For repeated freeze-thaw stability.

[0045] Figure 23 The traceability of the determination results of the standard reference for α-Syn protein substance solution. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention.

[0047] Example 1: Bioinformatics Analysis and Gene Cloning of α-Synuclein

[0048] According to publicly available information in the NCBI database, α-synuclein (α-Syn, also known as NACP140) is encoded by the SNCA gene (NG_011851.1) located on human chromosome 4q22.1, and is a small protein composed of 140 amino acids. The SNCA gene generates at least four major transcriptomic variants through alternative splicing, translating into α-Syn protein isoforms of varying lengths. Transcriptomic variant 1 is the most prevalent and abundant form, encoding the full-length 140-amino acid α-Syn. The certified full-length α-synuclein protein in the UniProt database is numbered P37840. Figure 1 As shown, the full-length α-synuclein sequence consists of three functional domains: an N-terminal amphiphilic region (amino acids 1-60), a hydrophobic intermediate region (amino acids 61-95), and an acidic C-terminal region (amino acids 96-140), with a molecular weight of approximately 14.46 kDa.

[0049] The coding gene for human α-Syn was cloned into the prokaryotic expression vector pET22b, which contains a T7 promoter and a histidine tag. The recombinant plasmid was then sequenced in its entirety to ensure complete sequence consistency with the reference sequence and the absence of any unexpected mutations. Figure 2As shown, the final expressed recombinant protein contains the MHHHHHH sequence (containing a methionine initiation residue and 6 histidine residues) introduced by the expression vector at its N-terminus, with a total length of 147 amino acids. The amino acid sequence of the recombinant protein is shown in SEQ ID NO:1. The introduction of the histidine tag allows for specific capture of the target protein via metal chelate affinity chromatography. Furthermore, the tag's location at the N-terminus does not affect the immunogenicity of the α-Syn C-terminal acidic region, thus preserving the recognition ability of standard substances for commonly used antibodies.

[0050] The technical considerations for choosing pET22b as the expression vector include: This vector carries a T7 promoter, providing strict induction control in the BL21(DE3) host bacterium, preventing leakage expression of the target protein in the uninduced state; the vector encodes a histidine tag of six consecutive His residues, a suitable length that provides sufficient binding force to the nickel column without excessively affecting the protein's physicochemical properties. The advantage of BL21(DE3) as an expression host lies in its lack of LON and OMPT proteases, reducing the risk of recombinant protein degradation by intracellular proteases. Low-temperature induction strategies (e.g., 16–20°C, 0.1–0.5 mmol / L IPTG, overnight expression) facilitate the accumulation of α-Syn in the cytoplasm in a soluble form, avoiding inclusion body formation and subsequent complex refolding processes, ensuring protein acquisition in its native conformation. Example 2: Expression, purification, and purity characterization of α-synuclein

[0051] 2.1 Protein Expression and Preliminary Purification

[0052] The validated recombinant plasmid was transformed into the *E. coli* expression strain BL21(DE3). Expression was induced by low-temperature isopropyl-β-D-thiogalactoside (IPTG). Since α-Syn is an inherently disordered protein with a small molecular weight (approximately 15 kDa), it exists in the cytoplasm of *E. coli* in a soluble form, requiring no inclusion body denaturation / renaturation treatment. After collecting the bacterial cells, they were sonicated and centrifuged to obtain the supernatant for subsequent purification.

[0053] The purification strategy employs a two-step chromatography method: The first step utilizes the specific binding of the recombinant protein's N-terminal histidine tag to a nickel-ion chelating resin (Ni-NTA) for affinity chromatography capture, followed by elution with gradient concentrations of imidazole to achieve initial enrichment of the target protein. The second step leverages the slightly acidic isoelectric point of α-Syn (theoretical pI approximately 4.67) to purify the protein using a DEAE anion exchange column under alkaline buffer conditions, removing charge variant impurities and residual host proteins. The design logic of the two-step purification strategy is as follows: affinity chromatography utilizes tag specificity to achieve rapid capture and enrichment, solving the efficiency problem of extracting the target protein from complex lysis buffers; ion exchange chromatography utilizes the inherent charge characteristics of the protein to achieve purification, addressing the removal of non-specifically adsorbed proteins and charge isomers that may remain after affinity chromatography.

[0054] The scientific basis for choosing the DEAE anion exchange column as the purification method is as follows: α-Syn has a theoretical isoelectric point of approximately 4.67. Under neutral to weakly alkaline buffer conditions (such as Tris-HCl buffer at pH 7.5 to 8.0), the protein carries a negative charge and can bind to the DEAE weak anion exchange resin. Elution is achieved by a linear salt gradient (such as 0 to 0.5 mol / L NaCl), based on the difference in surface charge density of the proteins. The C-terminal region (positions 96-140) of α-Syn is rich in acidic amino acids (Asp and Glu), giving the protein a unique charge characteristic that distinguishes its elution behavior from most host proteins, thus enabling effective separation. Even oxidized or deamidated forms of α-Syn, whose charge state differs slightly from the wild type, can be separated and removed by ion exchange chromatography, further ensuring the homogeneity of the standard raw materials.

[0055] 2.2 SDS-PAGE electrophoresis purity characterization

[0056] like Figure 3As shown, the purity of the purified α-Syn protein was characterized by SDS-PAGE electrophoresis. The electrophoresis conditions were as follows: 10 μg of α-Syn protein was mixed with an equal volume of 1X electrophoresis loading buffer, boiled in a water bath for 5 min, and 2 μg of α-Syn protein was loaded onto each lane. Separation was performed using a Bio-Rad Mini-PROTEAN precast gel at 120 V for 45 min. After electrophoresis, the sample was stained with Coomassie Brilliant Blue, destained with methanol-acetic acid solution, and then imaged. The electrophoresis results showed that the α-Syn protein lane had only one single, clear main band, with no obviously higher or lower molecular weight bands, indicating the absence of significant degradation products or high molecular weight aggregates in the sample. The electrophoresis results showed that the apparent molecular weight of the α-Syn protein was approximately 15 kD, consistent with the average molecular weight of 15.41 kD calculated based on its theoretical sequence. The SDS-PAGE results preliminarily confirmed the high purity and structural integrity of the purified product.

[0057] 2.3 Purity characterization by gel size exclusion chromatography

[0058] like Figure 4 As shown, the purity of the 1 mg / mL α-Syn protein standard was characterized by high-performance gel size exclusion liquid chromatography (SEC). The HPLC conditions were as follows: BioCore SEC-150 gel size exclusion column (7.8 mm × 300 mm); mobile phase: water:acetonitrile:trifluoroacetic acid (70:30:0.1, v:v:v); detection wavelength: 280 nm; flow rate: 0.5 mL / min; injection volume: 15 μL. The reason for choosing 280 nm as the detection wavelength is that the most likely impurities in α-Syn protein are other proteins and protein aggregates, and a wavelength of 280 nm ensures sufficient signal response for these proteinaceous organic compounds. The chromatogram of the 1 mg / mL sample shows that the impurity content of the α-Syn protein is extremely low, with a purity exceeding 99%.

[0059] like Figure 5 and Figure 6 As shown, to further confirm the impurity content, the sample concentration was increased to 5 mg / mL for SEC analysis. The main peak of the α-Syn protein was symmetrical and sharp, indicating high purity and sample homogeneity. Based on the detection results of the buffer blank, the peak after 12 min was the inorganic salt peak in the buffer; therefore, the peak after 12 min was not included in the protein purity calculation. By amplifying the baseline ( Figure 6As can be seen, there is a short fluctuation before the main peak, which may be a trace impurity peak. Purity was calculated using the peak area normalization method, with three replicates yielding results of 99.12%, 99.36%, and 99.31%, respectively, with an average purity of 99.26% and a relative standard deviation (RSD) of 0.13%. SEC purity characterization results indicate that the purified α-Syn protein raw material exists uniformly in monomeric form, with extremely low levels of aggregates and degradation products.

[0060] 2.4 Purity Characterization by Reversed-Phase High-Performance Liquid Chromatography

[0061] like Figure 7 and Figure 8 As shown in Table 1, to verify purity from different separation mechanisms, reversed-phase high-performance liquid chromatography (RP-HPLC) was used to characterize the 5 mg / mL α-Syn protein raw material. The chromatographic conditions were as follows: ZORBAX C8 column (4.6 mm × 250 mm); mobile phase A was 0.1% TFA-water, and mobile phase B was 0.1% TFA-acetonitrile; linear gradient elution was used; detection wavelength was 280 nm; flow rate was 0.5 mL / min; and injection volume was 15 μL. The gradient elution program was as follows: 0 to 5 min, holding at 80% A / 20% B; 5 to 35 min, linear gradient to 30% A / 70% B; 35 to 36 min, rapid transition to 0% A / 100% B and holding for 40 min; then, restoration to initial conditions for equilibration to 50 min.

[0062] Table 1 Gradient elution program for reversed-phase high-performance liquid chromatography

[0063]

[0064] As shown in Table 1, the core idea of ​​this gradient program design is as follows: isocratic elution for the first 5 minutes is used to stabilize the baseline and elute polar impurities; a 30-minute linear gradient from 5 to 35 minutes is used to separate the α-Syn protein main peak from possible hydrophobic impurities; and a high organic phase wash from 36 to 40 minutes ensures no residual protein remains on the column. The separation mechanism of reversed-phase chromatography is based on the hydrophobic interaction between the protein and the C8 stationary phase, which complements the molecular sieve separation mechanism of SEC.

[0065] C8 column analysis results ( Figure 7 This indicates that the purity of the α-Syn protein standard material remained above 99% under reversed-phase chromatography, consistent with the SEC analysis results. (From the magnified partial image...) Figure 8As can be seen from the data, there are two small peaks between 15 and 20 min compared to the buffer blank spectrum, which may be trace impurity peaks. Purity was calculated using the peak area normalization method, with three replicates yielding results of 99.43%, 99.42%, and 99.11%, respectively. The RP-HPLC results further confirmed the high purity of the α-Syn protein raw material.

[0066] Based on the purity characterization results of both SEC and RP-HPLC orthogonal separation modes, the final purity of the α-Syn protein standard raw material was determined to be 99.26% (normalized average of the three peak areas of SEC), with an RSD of 0.13%. The consistent high purity obtained from both separation mechanisms fully demonstrates the reliability of the purified product.

[0067] The positive and complementary characteristics of the three purity characterization methods are worth emphasizing: SDS-PAGE, based on molecular weight separation, mainly detects protein-level impurities and degradation products; SEC, based on molecular size and shape separation, can distinguish monomers, dimers, and high molecular weight aggregates; and RP-HPLC, based on hydrophobicity differences, can detect subtle differences in hydrophobicity caused by amino acid modifications or conformational variations. These three methods independently evaluate protein purity from different physicochemical dimensions. Impurities missed by any one method may be detected by the others, thus constituting a multidimensional cross-validation of the purity results.

[0068] Example 3: Protein molecular weight and mass spectrometry identification

[0069] like Figure 9 As shown, the purified α-Syn protein was identified by enzyme digestion using matrix-assisted laser-induced desorption / desorption-time-of-flight mass spectrometry (MALDI-TOF). Approximately 100 μg of α-Syn protein was added to urea at a final concentration of 8 mol / L and dithiothreitol (DTT) at a final concentration of 20 mmol / L. After mixing, the mixture was incubated at 37°C for 3 hours for denaturation and reduction. Iodoacetamide (IAA) at a final concentration of 40 mmol / L was added, and the mixture was incubated at room temperature in the dark for 30 minutes. 50 mmol / L ammonium bicarbonate was added to dilute the urea concentration to below 1 mol / L, and 2 μg of trypsin solution was added at an enzyme-to-protein ratio of 1:50. The mixture was incubated at 37°C for 18 hours. After the reaction was complete, 1% formic acid was added to terminate the reaction. The protein was desalted using a solid-phase extraction column, concentrated under vacuum, dried, and reconstituted with 0.1% formic acid-water. The resulting solution was mixed with an equal volume of saturated α-cyano-4-hydroxycinnamic acid matrix solution and then spotted onto a TLC plate for MALDI-TOF analysis.

[0070] A search of the MASCOT database for the digested α-Syn protein sequence revealed that α-Syn protein had the highest score, confirming the reliability of the results through statistical analysis. The identified peptide coverage was 95% (140 / 147 amino acid residues were identified), fully demonstrating the sequence correctness of the recombinant expression product.

[0071] Table 2. Identification results of MALDI-TOF enzyme-digested peptides

[0072]

[0073] As shown in Table 2, MALDI-TOF enzyme digestion identification covered multiple key regions of the protein sequence, detecting peptides from the N-terminal histidine tag region (positions 1-13) to the C-terminal acidic region (positions 110-147). The deviations between the measured molecular weights and theoretical values ​​of each peptide were within acceptable limits. In particular, the identification of the N-terminal peptide MHHHHHHMDVFMK confirmed the complete presence of the histidine tag, and the identification of the C-terminal peptide confirmed the integrity of the protein sequence, with no C-terminal truncation. The 95% sequence coverage indicates that only a very small number of short peptides were not detected by mass spectrometry. This is usually due to the loss of small molecular weight peptides during desalting or the weak signal in mass spectrometry detection, and does not affect the reliability of protein identification.

[0074] The significance of molecular weight mass spectrometry characterization lies not only in confirming sequence correctness, but more importantly in ruling out post-translational modifications that may occur during protein expression and purification: the minimal deviation (0.17 Da) between the ESI-MS measured molecular weight of 15414.03 Da and the theoretical value of 15414.20 Da excludes the presence of common modifications such as phosphorylation (+80 Da), acetylation (+42 Da), and oxidation (+16 Da), confirming that the recombinant protein exists in an unmodified form, which is crucial for standard substances used as calibration standards.

[0075] like Figure 10 As shown, the α-Syn protein sample was further characterized by high-resolution electrospray ionization mass spectrometry (ESI-MS). 5 μL of 1 mg / mL α-Syn protein sample was diluted 10-fold with pure water, and 10 μL was analyzed using a Thermo Fisher Q Exactive high-resolution mass spectrometer in ESI positive ion mode. The mass spectrum showed typical multi-charge envelope peaks, with charge states ranging from +10 to +21 valences. Deconvolution calculations determined the molecular weight of the α-Syn protein to be 15414.03 Da, a deviation of only 0.17 Da (approximately 11 ppm) from the theoretical molecular weight of 15414.20 Da, confirming the molecular integrity of the recombinant protein.

[0076] Example 4: Preparation and Dispensing of Standard Reference Materials

[0077] like Figure 11 As shown, the purified α-Syn protein standard material was diluted with distilled water to a concentration of 0.15 mg / mL according to the nominal concentration. Then, it was aliquoted into 500 μL screw-cap cryovials, 100 μL per tube, for a total of 200 tubes, and stored at -80°C. The entire aliquoting process was performed on ice to prevent protein degradation or aggregation. The nominal concentration of 0.15 mg / mL and the aliquot volume of 100 μL were chosen because: this concentration level matches the commonly used concentration range of α-Syn working standards in clinical testing, facilitating on-demand dilution; and the 100 μL volume meets the minimum sampling requirement for isotope dilution mass spectrometry while avoiding the need for repeated freeze-thaw cycles (single-use).

[0078] Example 5: Establishment of a fixed-value method – Optimization of hydrolysis time

[0079] For standard reference materials of pure proteins in solution, this invention employs isotope dilution mass spectrometry (AAA-IDMS) based on amino acid analysis for value determination. The core principle of this method is to completely hydrolyze the protein into free amino acids under high-temperature, strongly acidic conditions, and then infer the protein concentration by measuring the accurate content of specific free amino acids. Since amino acid composition is an inherent chemical property of proteins and is unaffected by protein conformation, aggregation state, or solution environment, this method can provide structure-independent absolute quantitative results and is an internationally recognized method for determining the value of primary protein standard reference materials.

[0080] like Figure 12 As shown, the hydrolysis time was first optimized. 20 µg of diluted α-Syn protein solution standard and 20 µg of the corresponding isotope-labeled amino acid mixture were weighed into 2 mL ampoules. After vacuum centrifugation at 55°C, 500 μL of 6 mol / L hydrochloric acid solution was added to each ampoule. After nitrogen purging for 2 min to remove oxygen, the ampoules were sealed and placed in a 110°C oven for hydrolysis. Six time points were set: 0, 16, 24, 48, 60, and 72 hours. After hydrolysis, the ampoules were dried using a nitrogen blower, reconstituted with 0.5 mL of 0.01 mol / L hydrochloric acid aqueous solution, filtered through a 0.22 μm filter membrane, and then analyzed.

[0081] This invention selects valine (Val), phenylalanine (Phe), and isoleucine (Ile), which are relatively stable under acid hydrolysis conditions, as quantitative targets. The scientific basis for choosing these three amino acids is: (a) their side chains are hydrophobic aliphatic or aromatic groups, and they do not undergo significant oxidative degradation under high-temperature hydrolysis with 6 mol / L hydrochloric acid (unlike sulfur-containing or indole amino acids such as methionine, cysteine, and tryptophan), nor do they undergo significant deamidation reactions (unlike asparagine and glutamine); (b) their abundance in α-Syn protein sequences is moderate (19 Val, 2 Phe, and 3 Ile), providing sufficient mass spectrometry signals. The peak area ratio of each amino acid to each labeled amino acid in the mass spectrum is used as a reference value; the hydrolysis is considered complete when the peak area ratio reaches its maximum or tends to a stable plateau. Figure 12 As shown, the results indicate that the peak area ratios of the three amino acids in the protein sample reached a stable plateau after 60 hours of hydrolysis. Therefore, 60 hours was determined to be the optimal hydrolysis time for this protein, and it was used for subsequent IDMS quantitative analysis. The significance of optimizing the hydrolysis time is that too short a hydrolysis time leads to incomplete protein decomposition and lower quantification; too long a hydrolysis time leads to degradation of sensitive amino acids, introducing additional errors. The optimal time of 60 hours is precisely the balance point between the two.

[0082] Example 6 Uniformity Test The homogeneity of the standard reference material is a key parameter for evaluating its intra-batch consistency. Based on the number of units dispensed (200 tubes), 11 tubes were randomly selected from the initial, middle, and final stages of dispensing, and labeled as 1 to 11, respectively. Each sample was analyzed using isotope dilution mass spectrometry based on amino acid analysis under controlled experimental conditions. A minimum sample size of 15 μL was selected, and each tube was analyzed three times.

[0083] Table 3. Homogeneity test data of α-Syn protein solution standard material (μg / g)

[0084]

[0085] As shown in Table 3, the statistical analysis of the homogeneity test data was performed using one-way ANOVA. The inter-group variation Q1 was 85.01 (degrees of freedom V1=10), and the within-group variation Q2 was 430.58 (degrees of freedom V2=22). The calculated F-value was 0.434, which is much smaller than the critical F-value F0.05(10,22)=2.30. The F-test results indicate that the differences between tubes are not significant, meaning the standard material has good homogeneity, and the dispensing process did not introduce significant intra-batch heterogeneity. The total coefficient of variation (CV) for the homogeneity test was 2.38%, meeting the homogeneity requirements for protein standard materials.

[0086] The uncertainty introduced by homogeneity was calculated according to JJF 1343. Since the within-group variance is greater than the between-group variance, the standard uncertainty introduced by homogeneity is 1.4 μg / g, and the corresponding relative uncertainty is 0.75%. This uncertainty will be included in the final combined uncertainty assessment.

[0087] Example 7 Stability Study

[0088] Table 4. Short-term stability study of α-Syn protein solution standard at 4°C (μg / g)

[0089]

[0090] As shown in Table 4 and Figure 13 As shown, after placing the standard substance at 4°C for 7 days, the protein content calculated by amino acid analysis remained essentially unchanged, and the slope of the t-test was not significant (p>0.05), indicating that the data were stable under 4°C conditions. Meanwhile, as... Figure 14 As shown in Table 5, the peak area of ​​the chromatogram did not change significantly within 7 days, further confirming the short-term stability under 4°C conditions.

[0091] Table 5. Monitoring of chromatographic peak area (mAU·min) of α-Syn protein solution standard at 4°C.

[0092]

[0093] As shown in Table 5, the chromatographic peak area fluctuated very little over 7 days at 4°C (from 60.0 to 61.4 mAU·min). The stability of the chromatographic peak area not only indicates that the total protein content remained unchanged (consistent with the amino acid analysis results), but more importantly, it proves that the protein has not degraded or aggregated (otherwise the peak area would have decreased or a new peak would have appeared). This dual verification strategy—amino acid analysis reflecting total stability and chromatographic peak area reflecting structural stability—constitutes a comprehensive evaluation of the stability of the standard substance.

[0094] 7.2 Short-term stability at room temperature

[0095] Table 6. Short-term stability study of α-Syn protein solution standard at room temperature (μg / g)

[0096]

[0097] As shown in Table 6 and Figure 15 As shown, the protein content remained essentially unchanged at room temperature for 7 days, and the slope was not significant according to the t-test. Figure 16 and Figure 17As shown, a linear fit was performed with time as the x-axis and normalized content as the y-axis, yielding a slope k = -0.0009861, an intercept b = 1.0003, and a very small standard deviation (s = 0.002278). The uncertainty introduced by short-term stability is uS = 0.27%. Room temperature stability verifies that short-term exposure to room temperature during transportation does not lead to significant changes in the value of the standard substance.

[0098] 7.3 Short-term stability at 40°C

[0099] Table 7. Short-term stability study of α-Syn protein solution standard at 40°C (μg / g)

[0100]

[0101] As shown in Table 7 and Figure 18 As shown, although the slope of the protein content after 7 days at 40°C was not significant according to the t-test, it was as follows: Figure 19 As shown, the peak area decreased on day 7 (from 60.4 to 56.3 mAU·min), suggesting that the protein may have begun to degrade. Amino acid analysis did not detect any changes because the degraded fragments still contain amino acids that can be released through hydrolysis, while chromatography sensitively reflects changes in protein integrity. Therefore, it is not recommended to store α-Syn protein solution standards at 40°C. This finding also highlights the necessity of a dual validation strategy (amino acid analysis + chromatographic monitoring): a single method may miss certain types of unstable signals.

[0102] 7.4 Long-term stability assessment like Figure 20 As shown, after two months of storage at -80°C, the protein content calculated from amino acids was 185.0 μg / g, essentially unchanged from the initial value. Simultaneously, chromatographic peak area monitoring showed peak areas of 62.1, 61.7, and 62.7 mAU·min in three repeated measurements, with no significant changes. The chromatograms also clearly show that the peak area remained essentially unchanged over two months. Therefore, the long-term stability of the α-Syn protein solution standard at -80°C is tentatively set at two months, and the research institution will continue to monitor it.

[0103] 7.5 Stability Study of Remelting

[0104] like Figure 21 and Figure 22 As shown in Table 8, to verify the remelting stability of the standard substance, it was subjected to four freeze-thaw cycles from -80°C to room temperature, with an injection volume of 15 μL each time for liquid chromatography analysis. The peak area decreased with the number of freeze-thaw cycles, as shown in Table 8.

[0105] Table 8 Freeze-thaw stability of α-Syn protein solution standard material (mAU·min)

[0106]

[0107] As shown in Table 8, the peak area remained stable after 1 to 2 freeze-thaw cycles (all at 63.9 mAU·min), but decreased to 62.4 mAU·min after 3 cycles, and further decreased to 61.9 mAU·min after 4 cycles. The t-statistic test showed |t|=3.87 > t-critical value=2.92, and the slope was significantly non-zero, indicating that the freeze-thaw process has a significant impact on protein integrity. As an inherently disordered protein, α-Syn may be partially aggregated or degraded due to the mechanical stress generated by ice crystal formation and melting during repeated freeze-thaw cycles. Therefore, this standard material is not recommended for repeated freeze-thaw cycles and should be used immediately after a single thawing.

[0108] The results of the freeze-thaw stability study have significant practical guiding significance for the use of reference materials. From a mechanistic perspective, the instability of α-Syn, as an inherently disordered protein, during the freeze-thaw process may stem from the following factors: during ice crystal formation, protein molecules are squeezed into the concentrated liquid phase region, and the increased local concentration induces enhanced protein-protein interactions; surface effects at the ice-liquid interface may promote partial folding or aggregation of proteins; repeated temperature changes cause protein molecules to undergo multiple conformational changes, increasing the probability of irreversible aggregation. This finding also explains why this invention chose a design of dispensing only 100 μL per tube—ensuring that each tube is a single-use quantity, fundamentally avoiding freeze-thaw problems.

[0109] Based on the above stability test results, the following recommendations are made for the use of α-Syn protein solution standard material: long-term storage at -80°C; cold chain transportation (can be stored for a short period of 7 days at 4°C); operation should be performed on an ice bath; use immediately after preparation; repeated freeze-thaw cycles are not recommended.

[0110] Example 8: Standard Reference Material Determination

[0111] 8.1 Fixed Value Method

[0112] The established isotope dilution mass spectrometry method based on amino acid analysis was used to determine the values ​​of α-Syn protein solution standards. Two researchers each extracted five units, and each unit was analyzed three times. HPLC-IDMS analysis was performed in an Agilent 1200 liquid chromatography system tandem with an AB5500 triple quadrupole mass spectrometer, using multiple reaction monitoring (MRM) mode. The chromatographic column was a Phenomenology Kinex C18 column (2.6 μm × 150 mm × 2.1 mm); elution conditions: mobile phase A (water) containing 0.1% trifluoroacetic acid and mobile phase B (100% acetonitrile) were eluted at a 91:9 volume ratio with an isotropic gradient for 10 min at a flow rate of 200 μL / min.

[0113] Table 9. HPLC-IDMS Analytical Mass Spectrometry Acquisition Conditions

[0114]

[0115] As shown in Table 9, the mass spectrometry parameters have been systematically optimized, mainly including ionization conditions, declustering voltage and inlet voltage for precursor ion scanning, and collision energy generated by daughter ions. The core advantage of the MRM mode lies in achieving high specificity detection through dual selection of precursor and daughter ions, effectively eliminating matrix interference. The selection principle for monitored ion pairs is to select the fragment ion pair with the highest signal intensity and the lowest background interference as the quantitative ion pair.

[0116] The constant value is calculated using the bracket method, and the formula is as follows: C = P × mstandard × (Rsample - R2) × (W1 - R1 × W1) / [M × (R1 - R2)] Where C represents the concentration of amino acids after sample hydrolysis; M represents the mass of the sample in the hydrolysis solution; R1 represents the peak area ratio of amino acids to labeled amino acids in the high-standard solution; R2 represents the peak area ratio of amino acids to labeled amino acids in the low-standard solution; P represents the purity of amino acids; mstandard represents the mass of labeled amino acids in the hydrolyzed sample; Rsample represents the peak area ratio of amino acids to labeled amino acids after sample hydrolysis; and W1 represents the mass ratio of amino acids to labeled amino acids in the high-standard solution. The core advantage of the bracket method lies in eliminating the influence of matrix effects and instrument drift on the quantitative results by sandwiching the response value of the sample under test between the high-standard and low-standard calibration points.

[0117] 8.2 Fixed Value Data and Statistical Analysis

[0118] Table 10. Standard reference values ​​for α-Syn protein solution (μg / g)

[0119]

[0120] As shown in Table 10, the results obtained by the two operators, each taking 5 units and analyzing each unit 3 times, showed good consistency. The average value for operator 1 was 186.3 μg / g (CV = 2.0%), and the average value for operator 2 was 185.2 μg / g (CV = 1.9%). The coefficients of variation for both sets of data were less than 3%, indicating excellent reproducibility of the method.

[0121] Triple statistical tests were performed on the setpoint data from the two operators in turn: (1) Outlier testing: The Dixon method and Grubbs method were used for testing. In the Dixon method, r1=0.127 and rn=0.078 for operator 1, and r1=0.084 and rn=0.176 for operator 2, both less than the critical value f(0.05,15)=0.565, indicating no outliers. In the Grubbs method, the maximum value of |V| / S for operator 1 was 1.859, and for operator 2 it was 1.831, both less than the critical value g2.41, indicating all data were retained. The application of the dual outlier testing method ensured that it was not affected by the bias of a single statistical method.

[0122] (2) Normality test: The Shapiro-Wilke test was used. The W value of operator 1 was 0.9659 and the W value of operator 2 was 0.9295. Both fell within the acceptance interval of W(15,0.95)=[0.8815,1], indicating that the two sets of data were normally distributed. Normality is a prerequisite assumption for the subsequent t-test.

[0123] (3) Test of mean consistency: A t-test was performed on the two sets of fixed-value data. At a significance level of α=0.05, the p-value was 0.39>0.05, indicating that there was no significant difference in the mean values ​​of the two sets of data. This shows that the two operators have comparable skill levels, and the inter-operator variation in the fixed-value method is negligible.

[0124] Independent dual-operation value setting is an internationally accepted practice in the development of protein standard materials, aiming to verify the robustness of the value setting method. In this invention, the difference in the average values ​​between the two operators was only 1.1 μg / g (186.3 vs. 185.2), a relative difference of 0.59%, far less than the method's coefficient of variation (approximately 2%), indicating that the value setting method is not significantly affected by differences in individual operator habits. Simultaneously, a four-fold statistical test (Dixon method + Grubbs method outlier test → Shapiro-Wilke normality test → t-test for consistency) forms a progressive data quality assurance system: first, confirming the absence of outlier interference; second, confirming that the data distribution meets the assumptions for subsequent parameter tests; and finally, confirming the consistency between the two independent results.

[0125] Based on the above statistical test results, the average value of the two operators' determination results was taken as the determination value of the standard substance, and the final determination result was 186.7 μg / g.

[0126] Example 9 Uncertainty Assessment

[0127] Table 11 Uncertainty Analysis Based on IDMS Values ​​from Amino Acid Analysis

[0128]

[0129] As shown in Table 11, the uncertainties of the determination method mainly fall into two categories: Type A and Type B. Type A uncertainties consist of method reproducibility (1.9%) and protein purity determination (0.13%), obtained through statistical analysis of actual repeated measurement data. Type B uncertainties include uncertainties introduced by balance weighing, uncertainties in the purity of amino acid standard substances, and uncertainties in the estimation of hydrolysis efficiency, all assessed based on information provided by equipment specifications or standard substance certificates.

[0130] Among the various uncertainties, method reproducibility (1.9%) and Ile standard substance purity (1.5%) are the two largest contributing components, together accounting for the dominant position of the total uncertainty. The uncertainty introduced by balance weighing (10⁻³ to...) The contribution of the mass (in milligrams) to the total uncertainty is negligible because the weighed mass (in milligrams) is much greater than the balance precision (0.01 mg). The uncertainty of hydrolysis efficiency (1.0%) is estimated based on international comparative empirical values, reflecting the unavoidable trace amino acid loss or incomplete hydrolysis during acid hydrolysis.

[0131] The uncertainty of the determination method (2.7%), the uniformity uncertainty (0.75%), and the short-term stability uncertainty (0.27%) were synthesized using the root sum of squares formula, yielding a combined standard uncertainty of uC = 2.8%. Taking a coverage factor k = 2 (corresponding to a 95% confidence level), the expanded uncertainty U = 5.6%. Therefore, the final determination result for the standard material is (186.7 ± 5.3) μg / g (k = 2, confidence level approximately 95%). This uncertainty level is comparable to that of similar primary protein standard materials internationally.

[0132] Among the sources of uncertainty, method reproducibility and the purity of amino acid reference materials are the two largest contributors. Ways to reduce the uncertainty of method reproducibility include increasing the number of repeated measurements and optimizing the uniformity of hydrolysis conditions; reducing the uncertainty of amino acid reference material purity depends on obtaining higher purity amino acid reference materials. The uncertainty introduced by balance weighing contributes very little to the total uncertainty because this method uses a weighing method rather than a volumetric method to measure samples and standards. The inherent high precision of the weighing method is an important basis for ensuring the accuracy of the determination. The uncertainty of hydrolysis efficiency reflects that under acid hydrolysis conditions at 110℃, some relatively stable peptide bonds (such as adjacent peptide bonds of branched chain amino acids like Val-Ile and Ile-Val) may require a longer time to completely break. The optimized hydrolysis time of 60 hours has minimized this impact.

[0133] Example 10: Determination of Standard Material Density and Metrological Traceability

[0134] Table 12 Density determination data of α-Syn protein solution standard material

[0135]

[0136] As shown in Table 12, five parallel weighings were performed on each of the distilled water and α-Syn protein solution using a pipette (scale adjusted to 20 μL). The density of the distilled water at a measurement temperature of 20.9°C was 0.998013 g / cm³. The actual volume was calculated from the weighed mass of the distilled water and the density from the table. Then, based on this volume and the weighed mass of the α-Syn protein solution, the density of the α-Syn protein solution was calculated to be 0.966825 g / cm³. This density data can be used to convert between mass concentration (μg / g) and volume concentration (μg / mL).

[0137] like Figure 23 As shown, the metrological traceability chain of this reference material is: α-Syn protein reference material → National amino acid primary reference material → SI unit. The National amino acid primary reference material is valued using quantitative nuclear magnetic resonance and mass balance methods, and its value is traceable to the SI unit. This reference material is valued using isotope dilution mass spectrometry, with the National amino acid primary reference material as the calibration standard, thus achieving complete traceability of the final result to the SI unit. The establishment of this traceability chain provides this reference material with an internationally recognized metrological basis, serving as a common reference for value transfer between different detection platforms.

[0138] Example 11: Standard Material Suitability Verification

[0139] The suitability of the standard substance values ​​was validated using an enzyme-linked immunosorbent assay (ELISA) kit to confirm that the standard substance is accurate not only at the mass spectrometry level but also suitable for terminal immunoassay platforms. Standard curves were prepared using the standard substance at gradient concentrations (0.625 to 40 ng / mL), and repeated detections were performed in two wells. The results are shown in Table 13.

[0140] Table 13 Results of ELISA Applicability Validation

[0141]

[0142] As shown in Table 13, the ELISA validation results indicate that the standard substance exhibits a good dose-response relationship within the concentration range of 0.625 to 40 ng / mL, and the OD value (OD-B column) after subtracting the blank increases monotonically with the concentration gradient. The consistency of duplicate assays in both wells is good (the difference between the two wells at each concentration point is <10%), indicating that the immunoreactivity of the standard substance is uniform and reliable. At the highest concentration point (40 ng / mL), the OD-B value reaches 2.030, indicating excellent binding efficiency between the protein and the detection antibody; at the lowest concentration point (0.625 ng / mL), a signal higher than the blank can still be detected (OD-B=0.081), indicating that the standard substance still has measurable immunomodulatory activity in the low concentration range. These validation results confirm that this standard substance is not only accurate at the stoichiometric level but also retains the natural immunogenic epitope of the α-Syn protein, enabling it to be correctly recognized and quantified by antibodies in commercial ELISA kits, and can be used as a calibration standard for immunoassay platforms.

[0143] It is particularly noteworthy that α-Syn protein, as an inherently disordered protein, may undergo conformational changes under different buffering and storage conditions. The successful ELISA suitability validation demonstrates that the preparation and storage conditions of this invention effectively maintain the integrity of the protein's immunogenic epitopes, and the presence of the histidine tag and cryopreservation did not adversely affect antibody recognition of the protein. Furthermore, the ELISA standard curve exhibits a typical S-type dose-response relationship at seven concentration levels, consistent with the expectations of the four-parameter logistic regression model, further validating that the immunological properties of the standard substance are consistent with those of the natural α-Syn protein.

[0144] The core innovation of this invention lies in the systematic integration of protein engineering preparation technology and absolute quantitative metrology methods into an end-to-end standard substance development system. Its technical logic chain is as follows: First, at the protein preparation level, α-Syn, as an inherently disordered protein, possesses unique physicochemical properties, lacking a stable tertiary structure, having a slightly acidic isoelectric point (pI approximately 4.67), and a relatively small molecular weight (approximately 15 kDa). These properties dictate the design of the purification strategy: histidine-tagged affinity chromatography utilizes the specific binding of the tag to metal ions for rapid capture, while anion exchange chromatography leverages the protein's inherent charge characteristics to achieve high purity. This two-step strategy complements each other from different physicochemical dimensions, ultimately achieving a purity of >99%.

[0145] Secondly, at the quantification level, traditional UV spectrophotometry relies on the UV absorption of aromatic amino acid residues in proteins. However, α-Syn contains only four tyrosine residues and two phenylalanine residues, lacking tryptophan residues, resulting in an extremely low extinction coefficient and sensitivity to solution environment. The amino acid hydrolysis-isotope dilution mass spectrometry method employed in this invention fundamentally bypasses this limitation: by completely hydrolyzing the protein into free amino acids, absolute quantification is performed at the amino acid level, eliminating the influence of protein conformation on the quantification results. Three acid-hydrolyzable stable amino acids (Val, Phe, and Ile) are selected as the quantification targets. Complete protein decomposition is ensured through 60-hour hydrolysis optimization, and matrix effects are eliminated through bracketing calculations, ultimately achieving complete traceability to SI units.

[0146] Finally, at the quality assurance level, homogeneity testing ensured the intra-batch consistency of the 200 tubes of standard material, multi-temperature stability studies determined suitable storage and transportation conditions, independent dual-person value determination combined with quadruple statistical testing ensured the metrological reliability of the certified values, and ELISA suitability verification confirmed the compatibility of the standard material with the terminal immunoassay platform. The entire quality evaluation system forms a complete closed loop from protein preparation to end-use.

[0147] From a methodological perspective, the AAA-IDMS quantification method established in this invention differs fundamentally from existing peptide-level MRM methods (such as the α-Syn candidate reference measurement procedure published by LGC Group in Analyst in 2024): peptide-level methods rely on the quantification of specific peptides after enzyme digestion, and their accuracy is affected by multiple factors including enzyme digestion efficiency, peptide selectivity, and matrix effects; while the amino acid-level method of this invention performs quantification at the total hydrolysis level, completely unaffected by enzyme digestion efficiency, quantifying the chemical composition of the protein rather than structural fragments, thus possessing higher methodological reliability and universality. This paradigm shift from "structure-dependent" to "composition-dependent" quantification is the core of the methodological innovation of this invention.

[0148] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing and determining the value of α-synuclein standard substances, characterized in that, Includes the following steps: Step 1, Protein preparation: The gene encoding α-synuclein is cloned into a prokaryotic expression vector for recombinant expression. Soluble components containing the α-synuclein are collected and purified by affinity chromatography and ion exchange chromatography to obtain α-synuclein raw material with a purity ≥99%. Step 2, Standard Substance Dispensing: The α-synuclein raw material is diluted to the target concentration and then dispensed and stored to obtain α-synuclein solution standard substance; Step 3, Value Assignment: The standard substance is assigned a value using isotope dilution mass spectrometry based on amino acid analysis. The value assignment method includes: mixing the standard substance with isotope-labeled amino acids and then performing acid hydrolysis. At least two free amino acids that are stable under acid hydrolysis conditions are used as quantitative targets. The peak area ratio of each amino acid to the corresponding isotope-labeled amino acid is determined by liquid chromatography-mass spectrometry. The mass concentration value of the standard substance is calculated. The mass concentration value is traced back to the International System of Units (SI) through the primary amino acid standard substance. Step 4: Quality Evaluation: Conduct homogeneity testing and stability assessment on the standard substance to confirm that its homogeneity and stability meet the technical specifications of the standard substance.

2. The method according to claim 1, characterized in that, In step one, the expression vector carries a histidine tag, the affinity chromatography is nickel ion metal chelate affinity chromatography, and the ion exchange chromatography is anion exchange chromatography.

3. The method according to claim 2, characterized in that, In step one, the anion exchange chromatography utilizes the acidic isoelectric point characteristics of α-synuclein and employs a DEAE column for purification to remove charge variant impurities.

4. The method according to claim 1, characterized in that, In step three, the stable free amino acid is selected from at least two of valine, phenylalanine, and isoleucine.

5. The method according to claim 4, characterized in that, In step three, the acid hydrolysis conditions are: hydrolysis in 6 mol / L hydrochloric acid solution at 108°C to 112°C for 50 to 70 hours.

6. The method according to claim 5, characterized in that, In step three, the isotope dilution mass spectrometry method uses the bracket method for quantitative calculation, and the matrix effect is eliminated by encapsulating the sample to be tested with high-standard and low-standard solutions.

7. The method according to claim 1, characterized in that, Step one also includes characterizing the purity of the purified α-synuclein raw material using at least two of the following methods: gel size exclusion chromatography, reversed-phase high-performance liquid chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

8. The method according to claim 1, characterized in that, In step four, the stability study includes short-term stability studies under at least two different temperature conditions, long-term stability studies under conditions ranging from -80°C to -60°C, and remelting stability studies.

9. The method according to claim 1, characterized in that, In step three, the setting of values ​​is performed by at least two independent operators, and the set value data are sequentially tested for outliers, normality, and consistency of average values.

10. The method according to claim 1, characterized in that, The amino acid sequence of the α-synuclein is shown in SEQ ID NO:1.

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